Methods for isolating novel antimicrobial agents from hypermutable mammalian cells

ABSTRACT

Dominant-negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. By introducing these genes into mammalian cells new cell lines with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation or introduction of mutations by chemical mutagens. These methods are useful for generating novel and highly active antimicrobial molecules as well as superior antimicrobial agents from pre-existing chemicals. These methods are also useful for generating cell lines expressing novel antimicrobials that are useful for pharmaceutical manufacturing.

FIELD OF THE INVENTION

The present invention is related to the area of antimicrobial agents andcellular production of those agents. In particular, it is related to thefield of identification of novel antimicrobial agents by placingmammalian cells under selection in the presence of the microbe.

BACKGROUND OF THE INVENTION

For as long as man has shared the planet with microorganisms there havebeen widespread outbreaks of infectious disease and subsequentwidespread mortality associated with it. Although microorganisms and manfrequently share a symbiotic relationship, microorganisms can, undersome conditions, lead to sickness and death. The discovery, wide use anddissemination of antibiotics to treat microbial infection in both humanand animal populations over the last one hundred or so years has donemuch to control, and in some instances, eradicate some microbes andassociated infectious disease. However, microbes have a strongpropensity to evolve and alter their genetic makeup when confronted withtoxic substances that place them under life and death selectivepressures. Therefore, emerging infectious diseases currently pose animportant public health problem in both developed as well as developingcountries. Not only have microbes evolved to evade and defeat currentantibiotic therapeutics, but also there are novel and previouslyunrecognized and/or characterized bacterial fungal, viral, and parasiticdiseases that have emerged within the past two decades. Sass, Curr.Opin. in Drug Discov. &Develop. 2000, 3(5):646-654.

Since the accidental discovery of a penicillin-producing mold by Flemingthere has been steady progress in synthesizing, isolating andcharacterizing new and more effective beta-lactam antibiotics. Inaddition to the great success of the beta-lactam family of antibiotics,the newer fluoroquinolones have a broad-spectrum of bactericidalactivity as well as excellent oral bio-availability, tissue penetrationand favorable safety and tolerability profiles. King et al., Am. Fam.Physician, 2000, 61, 2741-2748. A newly devised four-generationclassification of the quinolone drugs accounts for the expandedantimicrobial spectrum of the more recently introduced fluoroquinolonesand their clinical indications. The so-called first generation drugs,which include nalidixic acid, are capable of achieving minimal serumlevels. The second-generation quinolones, such as ciprofloxacin, have anincreased gram-negative and systemic activity. The third-generationdrugs comprise pharmaceuticals such as levofloxacin and are havesignificant and expanded action against gram-positive bacteria andatypical pathogens. Finally, the fourth-generation quinolone drugs,which, to date, only includes trovofloxacin, are highly active againstanaerobes in addition to the activity described for the third-generationdrugs. Furthermore, the quinolone class of anti-microbial drugs can bedivided based on their pharmacokinetic properties and bioavailability.

Mammalian epithelial surfaces are remarkable for their ability toprovide critical physiologic functions in the face of frequent microbialchallenges. The fact that these mucosal surfaces remain infection-freein the normal host suggests that highly effective mechanisms of hostdefense have evolved to protect these environmentally exposed tissues.Throughout the animal and plant kingdoms, endogenous genetically encodedantimicrobial peptides have been shown to be key elements in theresponse to epithelial compromise and microbial invasion. Zasloff, Curr.Opin. Immunol., 1992, 4, 3-7; and Bevins, Ciba Found. Symp., 1994, 186,250-69. In mammals, a variety of such peptides have been identified,including the well-characterized defensins and cathelicidins and others(andropin, magainin, tracheal antimicrobial peptide, and PR-39; seeBevins, Ciba Found. Symp., 1994, 186, 250-69 and references therein). Amajor source of these host defense molecules is circulating phagocyticleukocytes. However, more recently, it has been shown that residentepithelial cells of the skin and respiratory, alimentary, andgenitourinary tracts also synthesize and release antimicrobial peptides.Both in vitro and in vivo data support the hypothesis that thesemolecules are important contributors to intrinsic mucosal immunity.Alterations in their level of expression or biologic activity canpredispose the organism to microbial infection. Huttner et al., Pediatr.Res., 1999, 45, 785-94.

Across the evolutionary scale species from insects to mammals to plantsdefend themselves against invading pathogenic microorganisms byutilizing cationic antimicrobial peptides that rapidly kill microbeswithout exerting toxicity to the host. Physicochemical peptide-lipidinteractions provide attractive mechanisms for innate immunity asdiscussed below. Many of these peptides form cationic amphipathicsecondary structures, typically alpha-helices and beta-sheets, which canselectively interact with anionic bacterial membranes via electrostaticinteractions. Rapid, peptide-induced membrane permeabilization andsubsequent cellular lysis is the result. Matsuzaki, Biochim. Biophys.Acta, 1999, 1462, 1-10.

The primary structures of a large number of these host-defense peptideshave been determined. While there is no primary structure homology, thepeptides are characterized by a preponderance of cationic andhydrophobic amino acids. The secondary structures of many of thehost-defense peptides have been determined by a variety of techniques.Sitaram et al., Biochim, Biophys. Acta, 1999, 1462, 29-54. The acyclicpeptides tend to adopt helical conformation, especially in media of lowdielectric constant, whereas peptides with more than one disulfidebridge adopt beta-structures.

As described above, one reason for the rise in microbial drug resistanceto the first line antimicrobial therapies in standard use today is theinappropriate and over-use of prescription antibiotics. Althoughbacteria are the most common organisms to develop drug-resistance, thereare numerous examples of demonstrated resistance in fungi, viruses, andparasites. The development of a resistant phenotype is a complexphenomenon that involves an interaction of the microorganism, theenvironment, and the patient, separately as well as in combination.Sitaram et al., Biochim. Biophys. Acta, 1999, 1462, 29-54. Themicroorganism in question may develop resistance while under antibioticselection or it may be a characteristic of the microbe prior to exposureto a given agent. There are a number of mechanisms of resistance toantibiotics that have been described, including genes that encodeantibiotic resistance enzymes that are harbored on extrachromosomalplasmids as well as DNA elements (e.g. transposable elements) that canreside either extra-chromosomally or within the host genome.

Due to the ability of microorganisms to acquire the ability to developresistance to antibiotics there is a need to continually develop novelantibiotics. Traditional methods to develop novel antibiotics haveincluded medicinal chemistry approaches to modify existing antibiotics(Kang et al., Bioorg. Med. Chem. Lett., 2000, 10, 95-99) as well asisolation of antibiotics from new organisms (Alderson et al., Res.Microbiol., 1993, 144, 665-72). Each of these methods, however, haslimitations. The traditional medicinal chemistry approach entailsmodification of an existing molecule to impart a more effectiveactivity. The chemist makes a “best guess” as to which parts of themolecule to alter, must then devise a synthetic strategy, synthesize themolecule, and then have it tested. This approach is laborious, requireslarge numbers of medicinal chemists and frequently results in a moleculethat is lower in activity than the original antibiotic. The secondapproach, isolation of novel antimicrobial agents, requires screeninglarge numbers of diverse organisms for novel antimicrobial activity.Then, the activity must be isolated from the microorganism. This is nota small task, and frequently takes many years of hard work to isolatethe active molecule. Even after the molecule is identified, it may notbe possible for medicinal chemists to effectively devise a syntheticstrategy due to the complexity of the molecule. Furthermore, thesynthetic strategy must allow for a cost-effective synthesis. Therefore,a method that would allow for creation of more effective antibioticsfrom existing molecules or allow rapid isolation of novel antimicrobialagents is needed to combat the ever-growing list of antibiotic resistantorganisms. The present invention described herein is directed to the useof random genetic mutation of a cell to produce novel antibiotics byblocking the endogenous mismatch repair activity of a host cell. Thecell can be a mammalian cell that produces an antimicrobial agentnaturally, or a cell that is placed under selective pressure to obtain anovel antimicrobial molecule that attacks a specific microbe. Moreover,the invention describes methods for obtaining enhanced antimicrobialactivity of a cell line that produces an antimicrobial activity due torecombinant expression or as part of the innate capacity of the cell toharbor such activity.

In addition, the generation of genetically altered host cells that arecapable of secreting an antimicrobial activity, which can be protein ornon-protein based, will be valuable reagents for manufacturing theentity for clinical studies. An embodiment of the invention describedherein is directed to the creation of genetically altered host cellswith novel and/or increased antimicrobial production that are generatedby a method that interferes with the highly ubiquitous andphylogenetically conserved process of mismatch repair.

The present invention facilitates the generation of novel antimicrobialagents and the production of cell lines that express elevated levels ofantimicrobial activity. Advantages of the present invention are furtherdescribed in the examples and figures described herein.

SUMMARY OF THE INVENTION

One embodiment of the present invention provides a method for generatinggenetically altered mammalian cells and placing the cells under directmicrobial selection as a means to isolate novel antimicrobial agents.Another embodiment provides a method for identifying novelmicrobe-specific toxic molecules by altering the ability of the cell tocorrect natural defects that occur in the DNA during the process of DNAreplication. Interference with this process, called mismatch repair,leads to genetically dissimilar sibling cells. These geneticallydissimilar cells contain mutations, ranging from one mutation/genome totwo or more mutations/genome, offer a rich population of cells fromwhich to select for specific output traits, such as the novel ability toresist microbial insult. The genetically altered cell generated bymanipulation of the mismatch repair process is then incubated with amicrobe that is normally toxic to cells. Most of the cells will rapidlylose viability and die; however, a subset of resistant cells will havethe capacity to resist the microbial insult. These cells express amolecule, protein or non-protein in structure, that imbues anantimicrobial activity to the newly selected mammalian clones. Thesenewly created cells can be expanded in vitro and the new moleculeisolated and characterized by standard methods that are well describedit the art. The novel molecule(s) are then tested for their ability tokill or inhibit the growth of the microbe by standard microbial assaysthat are well described in the art. Finally, the novel cell linegenerated serves as an additional resource for large-scale production ofthe novel antimicrobial agent for use in clinical studies. The processesdescribed herein are applicable to any mammalian cell and any microbefor which an antibiotic agent is sought.

The invention provides methods for rendering mammalian cellshypermutable as a means to generate antimicrobial agents.

The invention also provides methods for generating genetically alteredcell lines that secrete enhanced amounts of a known or novelantimicrobial polypeptide.

The invention also provides methods for generating genetically alteredcell lines that secrete enhanced amounts of a known or novelantimicrobial non-polypeptide based molecule.

The invention also provides methods for generating genetically alteredcell lines that do not secrete enhanced amounts of an antimicrobialpeptide or non-peptide molecule but rather have a cell-surface activemolecule that detoxifies the microbe under test.

The invention also provides methods for producing an enhanced rate ofgenetic hypermutation in a mammalian cell and use of this as the basisto select for microbial-resistant cell lines.

The invention also provides methods of mutating a known antimicrobialencoding gene of interest in a mammalian cell as a means to obtain amolecule with enhanced bactericidal activity.

The invention also provides methods for creating genetically alteredantimicrobial molecules in vivo.

The invention also provides methods for creating novel antimicrobialmolecules from pre-existing antimicrobial molecules by altering theinnate enzymatic or binding ability of the molecules by altering themismatch repair system within the host mammalian cell.

The invention also provides methods for creating a novel anti-microbialpolypeptide or non-polypeptide based molecule that has the capacity tobind in an irreversible manner to a microbe and thereby block binding ofthe pathogenic microbe to a host target organism and result in loss ofviability of the microbe.

The invention also provides methods for creating a novel antimicrobialpolypeptide or non-polypeptide based small molecule that can blockmicrobial cell growth and/or survival.

The invention also provides methods for creating a novel antimicrobialpolypeptide or non-polypeptide based biochemical that are able toirreversibly bind to toxic chemicals produced by pathogenic microbes.

The invention also provides methods for creating genetically alteredantimicrobial molecules, either peptide of non-peptide based, that haveenhanced pharmacokinetic properties in host organisms.

The invention also provides methods for creating genetically alteredcell lines that manufacture an antimicrobial molecules, either peptideof non-peptide based, for use in large-scale production of theantimicrobial agent for clinical studies.

These and other aspects of the invention are described in theembodiments below. In one embodiment of the invention described, amethod for making a microbial-sensitive mammalian cell microbe resistantby rendering the cell line hypermutable is provided. A polynucleotideencoding a dominant negative allele of a mismatch repair gene isintroduced into an mammalian cell. The cell becomes hypermutable as aresult of the introduction of the gene.

In another embodiment of the invention, an isolated hypermutable cell isprovided. The cell comprises a dominant negative allele of a mismatchrepair gene. The cell exhibits an enhanced rate of hypermutation.

In another embodiment of the invention, an isolated hypermutable cell isprovided. The cell comprises a dominant negative allele of a mismatchrepair gene. The cell exhibits an enhanced rate of hypermutation. Thepopulations of cells generated by introduction of the mismatch repairgene are grown in the presence of microbes that are toxic to the wildtype non-mutant cells. Cells are selected that are resistant to themicrobe and the novel molecule(s) isolated and characterized forantimicrobial activity by standard methods well described in the art.

In another embodiment of the invention, an isolated hypermutable cell isdescribed to create a novel antimicrobial molecule from a pre-existingantimicrobial molecule by altering the innate enzymatic or bindingability of the molecule.

In another embodiment of the invention, a method of creating a novelantimicrobial polypeptide or non-polypeptide based molecule that has thecapacity to bind in an irreversible manner to a microbe and therebyblock binding of the pathogenic microbe to a host target organism andresult in loss of viability of the microbe.

In another embodiment of the invention, a method of creating a novelantimicrobial polypeptide or non-polypeptide based small molecule thatcan block microbial cell growth and/or survival is described.

In another embodiment of the invention, a method of creating a novelantimicrobial polypeptide or non-polypeptide based biochemical that areable to irreversibly bind to toxic chemicals produced by pathogenicmicrobes is described.

In another embodiment of the invention, a method is provided forintroducing a mutation into a known endogenous gene encoding for anantimicrobial polypeptide or a non-protein based antimicrobial moleculeas a means to create a more efficacious antimicrobial. A polynucleotideencoding a dominant negative allele of a mismatch repair gene isintroduced into a cell. The cell becomes hypermutable as a result of theintroduction of the gene. The cell further comprises an antimicrobialgene(s) of interest. The cell is grown and tested to determine whetherthe gene encoding for an antimicrobial is altered and whether the novelmolecule is more active by standard microbiology assays well known inthe art.

In another embodiment of the invention, a gene or genes encoding for anantimicrobial molecule is introduced into a mammalian cell host that ismismatch repair defective. The cell is grown, and then clones areanalyzed for enhanced antimicrobial characteristics.

In another embodiment of the invention, a method is provided forproducing new phenotypes of a cell. A polynucleotide encoding a dominantnegative allele of a mismatch repair gene is introduced into a cell. Thecell becomes hypermutable as a result of the introduction of the gene.The cell is grown and tested for the expression of new phenotypes wherethe phenotype is enhanced secretion of a novel or known antimicrobialpolypeptide.

In another embodiment of the invention, a method is provided forproducing new phenotypes of a cell. A polynucleotide encoding a dominantnegative allele of a mismatch repair gene is introduced into a cell. Thecell becomes hypermutable as a result of the introduction of the gene.The cell is grown and tested for the expression of new phenotypes wherethe phenotype is enhanced secretion of a novel or known antimicrobialnon-polypeptide based molecule.

In another embodiment of the invention, a method is provided forproducing new phenotypes of a cell. A polynucleotide encoding a dominantnegative allele of a mismatch repair gene is introduced into a cell. Thecell becomes hypermutable as a result of the introduction of the gene.The cell is grown and tested for the expression of new phenotypes wherethe phenotype is enhanced antimicrobial activity of a novel or knownantimicrobial polypeptide that is not secreted.

In another embodiment of the invention, a method is provided forproducing new phenotypes of a cell. A polynucleotide encoding a dominantnegative allele of a mismatch repair gene is introduced into a cell. Thecell becomes hypermutable as a result of the introduction of the gene.The cell is grown and tested for the expression of new phenotypes wherethe phenotype is enhanced antimicrobial activity of a novel or knownantimicrobial non-polypeptide based molecule that is not secreted.

In another embodiment of the invention, a method is provided forrestoring genetic stability in a cell containing a polynucleotideencoding a dominant negative allele of a mismatch repair gene. Theexpression of the dominant negative mismatch repair gene is suppressedand the cell is restored to its former genetic stability.

In another embodiment of the invention, a method is provided forrestoring genetic stability in a cell containing a polynucleotideencoding a dominant negative allele of a mismatch repair gene and anewly selected phenotype. The expression of the dominant negativemismatch repair gene is suppressed and the cell restores its geneticstability and the new phenotype is stable.

These and other embodiments of the invention provide the art withmethods that generate enhanced mutability in cells and animals as wellas providing cells and animals harboring potentially useful mutationsand novel protein and non-protein based molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representative in situ β-galactosidase staining ofTK-hPMS2-134 or TKvect cells to measure for cells containing geneticallyaltered β-galactosidase genes; arrows indicate Blue (β-galactosidasepositive) cells.

FIG. 2 is a schematic representation of sequence of alterations of theβ-galactosidase gene produced by expression of TK-hPMS2-134 host cellsin TK cells.

FIGS. 3A, 3B and 3C show a representative immunoprecipitation of invitro translated hPMS2 and hMLH1 proteins.

FIG. 4 shows representative complementation of MMR activity intransduced SH cells.

FIG. 5 is a representative photograph of Syrian hamster TK-ts13 cellstransfected with a eukaryotic expression vector that produces a novelanti-microbial polypeptide.

FIG. 6 is a representative graph showing TK-hPMS-134 transfected TKcells can suppress the growth of bacteria in vitro.

The presented invention is directed to, in part, methods for developinghypermutable mammalian cells by taking advantage of the conservedmismatch repair process of host cells. Mismatched repair process isdescribed in several references. Baker et al., Cell, 1995, 82, 309 319;Bronner et al., Nature, 1994, 368, 258 261; de Wind et al., Cell, 1995,82, 321 330; and Drummond et al., Science, 1995, 268, 1909 1912.Dominant negative alleles of such genes, when introduced into cells ortransgenic animals, increase the rate of spontaneous mutations byreducing the effectiveness of DNA repair and thereby render the cells oranimals hypermutable. Hypermutable cells or animals can then be utilizedto develop new mutations in a gene of interest or in a gene whosefunction has not been previously described. Blocking mismatch repair incells such as, for example, mammalian cells or mammalian cellstransfected with genes encoding for specific antimicrobial peptides ornon-peptide based antimicrobials, can enhance the rate of mutationwithin these cells leading to clones that have novel or enhancedantimicrobial activity or production and/or cells that containgenetically altered antimicrobials with enhanced biochemical activityagainst a range of opportunistic microbes.

The process of mismatch repair, also called mismatch proofreading, iscarried out by protein complexes in cells ranging from bacteria tomammalian cells. Modrich, Science, 1994, 266, 1959 1960. A mismatchrepair gene is a gene that encodes for one of the proteins of such amismatch repair complex. Baker et. al., Cell, 1995, 82, 309 319; Bronneret al., Nature, 1994, 368, 258 261; de Wind et al., Cell, 1995, 82, 321330; Drummond et al., Science, 1995, 268, 1909 1912; and Modrich,Science, 1994, 266, 1959 1960. Although not wanting to be bound by anyparticular theory of mechanism of action, a mismatch repair complex isbelieved to detect distortions of the DNA helix resulting fromnon-complementary pairing of nucleotide bases. The non-complementarybase on the newer DNA strand is excised, and the excised base isreplaced with the appropriate base that is complementary to the olderDNA strand. In this way, cells eliminate many mutations, which occur asa result of mistakes in DNA replication.

Dominant negative alleles cause a mismatch repair defective phenotypeeven in the presence of a wild-type allele in the same cell. An exampleof a dominant negative allele of a mismatch repair gene is the humangene hPMS2-134, which carries a truncation mutation at codon 134.Nicolaides et al., Mol. Cell. Biol., 1998, 18, 1635-1641. The mutationcauses the product of this gene to abnormally terminate at the positionof the 134th amino acid, resulting in a shortened polypeptide containingthe N-terminal 133 amino acids. Such a mutation causes an increase inthe rate of mutations that accumulate in cells after DNA replication.Expression of a dominant negative allele of a mismatch repair generesults in impairment of mismatch repair activity, even in the presenceof the wild-type allele. Any allele, which produces such effect, can beused in this invention.

Dominant negative alleles of a mismatch repair gene can be obtained fromthe ells of humans, animals, yeast, bacteria, or other organisms. Prollaet al., Science, 1994, 264, 1091 1093; Strand et al., Nature, 1993, 365,274 276; and Su et al., J. Biol. Chem., 1988, 263, 6829 6835. Screeningcells for defective mismatch repair activity can identify such alleles.Cells from animals or humans with cancer can be screened for defectivemismatch repair. Cells from colon cancer patients may be particularlyuseful. Parsons et al., Cell, 1993, 75, 1227 1236; and Papadopoulos etal., Science, 1993, 263, 1625 1629. Genomic DNA, cDNA, or mRNA from anycell encoding a mismatch repair protein can be analyzed for variationsfrom the wild type sequence. Perucho, Biol. Chem., 1996, 377, 675 684.Dominant negative alleles of a mismatch repair gene can also be createdartificially, for example, by producing variants of the hPMS2-134 alleleor other mismatch repair genes. Various techniques of site-directedmutagenesis can be used. The suitability of such alleles, whethernatural or artificial, for use in generating hypermutable cells oranimals can be evaluated by testing the mismatch repair activity causedby the allele in the presence of one or more wild-type alleles, todetermine if it is a dominant negative allele.

A cell or an animal into which a dominant negative allele of a mismatchrepair gene has been introduced will become hypermutable. This meansthat the spontaneous mutation rate of such cells or animals is elevatedcompared to cells or animals without such alleles. The degree ofelevation of the spontaneous mutation rate can be at least 2-fold,5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or1000-fold that of the normal cell or animal.

According to one aspect of the invention, a polynucleotide encoding adominant negative form of a mismatch repair protein is introduced into acell. The gene can be any dominant negative allele encoding a proteinthat is part of a mismatch repair complex, for example, PMS2, PMS1,MLH1, or MSH2. The dominant negative allele can be naturally occurringor made in the laboratory. The polynucleotide can be in the form ofgenomic DNA, cDNA, RNA, or a chemically synthesized polynucleotide.

The polynucleotide can be cloned into an expression vector containing aconstitutively active promoter segment (such as but not limited to CMV,SV40, Elongation Factor or LTR sequences) or to inducible promotersequences such as the steroid inducible pIND vector (InVitrogen), wherethe expression of the dominant negative mismatch repair gene can beregulated. The polynucleotide can be introduced into the cell bytransfection.

Transfection is any process whereby a polynucleotide is introduced intoa cell. The process of transfection can be carried out in a livinganimal, e.g., using a vector for gene therapy, or it can be carried outin vitro, e.g., using a suspension of one or more isolated cells inculture. The cell can be any type of eukaryotic cell, including, forexample, cells isolated from humans or other primates, mammals or othervertebrates, invertebrates, and single celled organisms such asprotozoa, yeast, or bacteria.

In general, transfection will be carried out using a suspension ofcells, or a single cell, but other methods can also be applied as longas a sufficient fraction of the treated cells or tissue incorporates thepolynucleotide so as to allow transfected cells to be grown andutilized. The protein product of the polynucleotide may be transientlyor stably expressed in the cell. Techniques for transfection are wellknown and available techniques for introducing polynucleotides includebut are not limited to electroporation, transduction, cell fusion, theuse of calcium chloride, and packaging of the polynucleotide togetherwith lipid for fusion with the cells of interest. Once a cell has beentransfected with the mismatch repair gene, the cell can be grown andreproduced in culture. If the transfection is stable, such that the geneis expressed at a consistent level for many cell generations, then acell line results.

An isolated cell is a cell obtained from a tissue of humans or animalsby mechanically separating out individual cells and transferring them toa suitable cell culture medium, either with or without pretreatment ofthe tissue with enzymes, e.g., collagenase or trypsin. Such isolatedcells are typically cultured in the absence of other types of cells.Cells selected for the introduction of a dominant negative allele of amismatch repair gene may be derived from a eukaryotic organism in theform of a primary cell culture or an immortalized cell line, or may bederived from suspensions of single-celled organisms.

The invention described herein is useful for creatingmicrobial-resistant mammalian cells that secrete new antimicrobialbiochemical agents, either protein or non-protein in nature.Furthermore, the invention can be applied to cell lines that expressknown antimicrobial agents as a means to enhance the biochemicalactivity of the antimicrobial agent.

Once a transfected cell line has been produced, it can be used togenerate new mutations in one or more gene(s) of interest or in genesthat have not been previously described. A gene of interest can be anygene naturally possessed by the cell line or introduced into the cellline by standard methods known in the art. An advantage of usingtransfected cells or to induce mutation(s) in a gene or genes ofinterest that encode antimicrobial activity is that the cell need not beexposed to mutagenic chemicals or radiation, which may have secondaryharmful effects, both on the object of the exposure and on the workers.Furthermore, it has been demonstrated that chemical and physicalmutagens are base pair specific in the way they alter the structure ofDNA; the invention described herein results in mutations that are notdependent upon the specific nucleotide or a specific string ofnucleotides and is a truly random genetic approach. Therefore, use ofthe present invention to obtain mutations in novel or knownantimicrobial genes will be much more efficient and have a higherlikelihood of success in contrast to conventional mutagenesis withchemical or irradiation. Once a new antimicrobial trait is identified ina sibling cell, the dominant negative allele can be removed from thecell by a variety of standard methods known in the art. For example, thegene can be directly knocked out the allele by technologies used bythose skilled in the art or use of a inducible expression system; thedominant-negative allele is driven by a standard promoter that isregulated by inclusion of an inducer, withdrawal of the inducer resultsin attenuation of the expression of the dominant negative mismatchrepair mutant and a normal DNA repair process will ensue.

New antimicrobial agents are selected from cells that have been exposedto the dominant negative mismatch repair process followed by incubatingthe mutant cells in the presence of the microbe for which an novelantimicrobial agent is sought. The novel antimicrobial agent is purifiedby standard methods known to those skilled in the art and characterized.The antimicrobial agents are re-screened to determine the specificactivity of the novel antimicrobial as well as tested against a broadrange of microbes to determine spectrum of activity. The gene(s) thatencode the novel antimicrobial are isolated by standard well knownmethods to those in the art. The mutations can be detected by analyzingfor alterations in the genotype of the cells by examining the sequenceof genomic DNA, cDNA, messenger RNA, or amino acids associated with thegene of interest. A mutant polypeptide can be detected by identifyingalterations in electrophoretic mobility, spectroscopic properties, orother physical or structural characteristics of a protein encoded by amutant gene when the cell that has undergone alteration encodes a knownantimicrobial that is altered by the means described in the currentinvention to obtain a more efficacious antimicrobial. Examples ofmismatch repair proteins and nucleic acid sequences include thefollowing: PMS2 (mouse) (SEQ ID NO: 7) MEQTEGVSTE CAKAIKPIDG KSVHQICSGQVILSLSTAVK 60 ELIENSVDAG ATTIDLRLKD YGVDLIEVSD NGCGVEEENF EGLALKHHTSKIQEEADLTQ 120 VETFGFRGEA LSSLCALSDV TISTCHGSAS VGTRLVFDHN GKITQKTPYPRPKGTTVSVQ 180 HLFYTLPVRY KEFQRNIKKE YSKMVQVLQA YCIISAGVRV SCTNQLGQGKRHAVVCTSGT 240 SGMKENIGSV FGQKQLQSLI PFVQLPPSDA VCEEYGLSTS GRHKTFSTFRASFHSARTAP 300 GGVQQTGSFS SSIRGPVTQQ RSLSLSMRFY HMYNRHQYPF VVLNVSVDSECVDINVTPDK 360 RQILLQEEKL LLAVLKTSLI GMFDSDANKL NVNQQPLLDV EGNLVKLHTAELEKPVPGKQ 420 DNSPSLKSTA DEKRVASISR LREAFSLHPT KEIKSRGPET AELTRSFPSEKRGVLSSYPS 480 DVISYRGLRG SQDKLVSPTD SPGDCMDREK IEKDSGLSST SAGSEEEFSTPEVASSFSSD 540 YNVSSLZDRP SQETINCGDL DCRPPGTGQS LKPEDHGYQC KALPLARLSPTNAKRFKTEE 600 RPSNVNISQR LPGPQSTSAA EVDVAIKMNK RIVLLEFSLS SLAKRMKQLQHLKAQNKHEL 660 SYRKFRAKIC PGENQAAEDE LRKEISKSMF AEMEILGQFN LGFIVTKLKEDLFLVDQHAA 720 DEKYNFEMLQ QHTVLQAQRL ITPQTLNLTA VNEAVLIENL EIFRKNGFDFVIDEDAPVTE 780 RAKLISLPTS KNWTFGPQDI DELIFMLSDS PGVMCRPSRV RQMFASRACRKSVMIGTALN 840 ASEMKKLITH MGEMDHPWNC PHGRPTMRHV ANLDVISQN 859 PMS2(mouse cDNA) (SEQ ID NO: 8) gaattccggt gaaggtcctg aagaatttcc agattcctga60 gtatcattgg aggagacaga taacctgtcg tcaggtaacg atggtgtata tgcaacagaa 120atgggtgttc ctggagacgc gtcttttccc gagagcggca ccgcaactct cccgcggtga 180ctgtgactgg aggagtcctg catccatgga gcaaaccgaa ggcgtgagta cagaatgtgc 240taaggccatc aagcctattg atgggaagtc agtccatcaa atttgttctg ggcaggtgat 300actcagttta agcaccgctg tgaaggagtt gatagaaaat agtgtagatg ctggtgctac 360tactattgat ctaaggctta aagactatgg ggtggacctc attgaagttt cagacaatgg 420atgtggggta gaagaagaaa actttgaagg tctagctctg aaacatcaca catctaagat 480tcaagagttt gccgacctca cgcaggttga aactttcggc tttcgggggg aagctctgag 540ctctctgtgt gcactaagtg atgtcactat atctacctgc cacgggtctg caagcgttgg 600gactcgactg gtgtttgacc ataatgggaa aatcacccag aaaactccct acccccgacc 660taaaggaacc acagtcagtg tgcagcactt attttataca ctacccgtgc gttacaaaga 720gtttcagagg aacattaaaa aggagtattc caaaatggtg caggtcttac aggcgtactg 780tatcatctca gcaggcgtcc gtgtaagctg cactaatcag ctcggacagg ggaagcggca 840cgctgtggtg tgcacaagcg gcacgtctgg catgaaggaa aatatcgggt ctgtgtttgg 900ccagaagcag ttgcaaagcc tcattccttt tgttcagctg ccccctagtg acgctgtgtg 960tgaagagtac ggcctgagca cttcaggacg ccacaaaacc ttttctacgt ttcgggcttc 1020atttcacagt gcacgcacgg cgccgggagg agtgcaacag acaggcagtt tttcttcatc 1080aatcagaggc cctgtgaccc agcaaaggtc tctaagcttg tcaatgaggt tttatcacat 1140gtataaccgg catcagtacc catttgtcgt ccttaacgtt tccgttgact cagaatgtgt 1200ggatattaat gtaactccag ataaaaggca aattctacta caagaagaga agctattgct 1260ggccgtttta aagacctcct tgataggaat gtttgacagt gatgcaaaca agcttaatgt 1320caaccagcag ccactgctag atgttgaagg taacttagta aagctgcata ctgcagaact 1380agaaaagcct gtgccaggaa agcaagataa ctctccttca ctgaagagca cagcagacga 1440gaaaagggta gcatccatct ccaggctgag agaggccttt tctcttcatc ctactaaaga 1500gatcaagtct aggggtccag agactgctga actgacacgg agttttccaa gtgagaaaag 1560gggcgtgtta tcctcttatc cttcagacgt catctcttac agaggcctcc gtggctcgca 1620ggacaaattg gtgagtccca cggacagccc tggtgactgt atggacagag agaaaataga 1680aaaagactca gggctcagca gcacctcagc tggctctgag gaagagttca gcaccccaga 1740agtggccagt agctttagca gtgactataa cgtgagctcc ctagaagaca gaccttctca 1800ggaaaccata aactgtggtg acctggactg ccgtcctcca ggtacaggac agtccttgaa 1860gccagaagac catggatatc aatgcaaagc tctacctcta gctcgtctgt cacccacaaa 1920tgccaagcgc ttcaagacag aggaaagacc ctcaaatgtc aacatttctc aaagattgcc 1980tggtcctcag agcacctcag cagctgaggt cgatgtagcc ataaaaatga ataagagaat 2040cgtgctcctc gagttctctc tgagttctct agctaagcga atgaagcagt tacagcacct 2100aaaggcgcag aacaaacatg aactgagtta cagaaaattt agggccaaga tttgccctgg 2160agaaaaccaa gcagcagaag atgaactcag aaaagagatt agtaaatcga tgtttgcaga 2220gatggagatc ttgggtcagt ttaacctggg atttatagta accaaactga aagaggacct 2280cttcctggtg gaccagcatg ctgcggatga gaagtacaac tttgagatgc tgcagcagca 2340cacggtgctc caggcgcaga ggctcatcac accccagact ctgaacttaa ctgctgtcaa 2400tgaagctgta ctgatagaaa atctggaaat attcagaaag aatggctttg actttgtcat 2460tgatgaggat gctccagtca ctgaaagggc taaattgatt tccttaccaa ctagtaaaaa 2520ctggaccttt ggaccccaag atatagatga actgatcttt atgttaagtg acagccctgg 2580ggtcatgtgc cggccctcac gagtcagaca gatgtttgct tccagagcct gtcggaagtc 2640agtgatgatt ggaacggcgc tcaatgcgag cgagatgaag aagctcatca cccacatggg 2700tgagatggac cacccctgga actgccccca cggcaggcca accatgaggc acgttgccaa 2760tctggatgtc atctctcaga actgacacac cccttgtagc atagagttta ttacagattg 2820ttcggtttgc aaagagaagg ttttaagtaa tctgattatc gttgtacaaa aattagcatg 2880ctgctttaat gtactggatc catttaaaag cagtgttaag gcaggcatga tggagtgttc 2940ctctagctca gctacttggg tgatccggtg ggagctcatg tgagcccagg actttgagac 3000cactccgagc cacattcatg agactcaatt caaggacaaa aaaaaaaaga tatttttgaa 3056gccttttaaa aaaaaa PMS2 (human) (SEQ ID NO: 9) MERAESSSTE PAKAIKPIDRKSVHQICSGQ VVLSLSTAVK 60 ELVENSLDAG ATNIDLKLKD YGVDLIEVSD NGCGVEEENFEGLTLKHHTS KIQEFADLTQ 120 VETFGFRGEA LSSLCALSDV TISTCHASAK VGTRLMFDHNGKIIQKTPYP RPRGTTVSVQ 180 QLFSTLPVRH KEFQRNIKKE YAKMVQVLHA YCIISAGIRVSCTNQLGQGK RQPVVCTGGS 240 PSIKENIGSV FGQKQLQSLI PFVQLPPSDS VCEEYGLSCSDALHNLFYIS GFISQCTHGV 300 GRSSTDRQFF FINRRPCDPA KVCRLVNEVY HMYNRHQYPFVVLNISVDSE CVDINVTPDK 360 RQILLQEEKL LLAVLKTSLI GMFDSDVNKL NVSQQPLLDVEGNLIKMHAA DLEKPMVEKQ 420 DQSPSLRTGE EKKDVSISRL REAFSLRHTT ENKPHSPKTPEPRRSPLGQK RGMLSSSTSG 480 AISDKGVLRP QKEAVSSSHG PSDPTDRAEV EKDSGHGSTSVDSEGFSIPD TGSHCSSEYA 540 ASSPGDRGSQ EHVDSQEKAP ETDDSFSDVD CHSNQEDTGCKFRVLPQPTN LATPNTKRFK 600 KEEILSSSDI CQKLVNTQDM SASQVDVAVK INKKVVPLDFSMSSLAKRIK QLHHFAQQSE 660 GEQNYRKFRA KICPGENQAA EDELRKEISK TMFAEMEIIGQFNLGFIITK LNEDIFIVDQ 720 HATDEKYNFE MLQQHTVLQG QRLIAPQTLN LTAVNEAVLIENLEIFRKNG FDFVIDENAP 780 VTERAKLISL PTSKNWTFGP QDVDELIFML SDSPGVMCRPSRVKQMFASR ACRKSVMIGT 840 ALNTSEMKKL ITHMGEMDHP WNCPHGRPTM RHIANLGVIS QN862 PMS2 (human cDNA) (SEQ ID NO: 10) cgaggcggat cgggtgttgc atccatggagcgagctgaga 60 gctcgagtac agaacctgct aaggccatca aacctattga tcggaagtcagtccatcaga 120 tttgctctgg gcaggtggta ctgagtctaa gcactgcggt aaaggagttagtagaaaaca 180 gtctggatgc tggtgccact aatattgatc taaagcttaa ggactatggagtggatctta 240 ttgaagtttc agacaatgga tgtggggtag aagaagaaaa cttcgaaggcttaactctga 300 aacatcacac atctaagatt caagagtttg ccgacctaac tcaggttgaaacttttggct 360 ttcgggggga agctctgagc tcactttgtg cactgagcga tgtcaccatttctacctgcc 420 acgcatcggc gaaggttgga actcgactga tgtttgatca caatgggaaaattatccaga 480 aaacccccta cccccgcccc agagggacca cagtcagcgt gcagcagttattttccacac 540 tacctgtgcg ccataaggaa tttcaaagga atattaagaa ggagtatgccaaaatggtcc 600 aggtcttaca tgcatactgt atcatttcag caggcatccg tgtaagttgcaccaatcagc 660 ttggacaagg aaaacgacag cctgtggtat gcacaggtgg aagccccagcataaaggaaa 720 atatcggctc tgtgtttggg cagaagcagt tgcaaagcct cattccttttgttcagctgc 780 cccctagtga ctccgtgtgt gaagagtacg gtttgagctg ttcggatgctctgcataatc 840 ttttttacat ctcaggtttc atttcacaat gcacgcatgg agttggaaggagttcaacag 900 acagacagtt tttctttatc aaccggcggc cttgtgaccc agcaaaggtctgcagactcg 960 tgaatgaggt ctaccacatg tataatcgac accagtatcc atttgttgttcttaacattt 1020 ctgttgattc agaatgcgtt gatatcaatg ttactccaga taaaaggcaaattttgctac 1080 aagaggaaaa gcttttgttg gcagttttaa agacctcttt gataggaatgtttgatagtg 1140 atgtcaacaa gctaaatgtc agtcagcagc cactgctgga tgttgaaggtaacttaataa 1200 aaatgcatgc agcggatttg gaaaagccca tggtagaaaa gcaggatcaatccccttcat 1260 taaggactgg agaagaaaaa aaagacgtgt ccatttccag actgcgagaggccttttctc 1320 ttcgtcacac aacagagaac aagcctcaca gcccaaagac tccagaaccaagaaggagcc 1380 ctctaggaca gaaaaggggt atgctgtctt ctagcacttc aggtgccatctctgacaaag 1440 gcgtcctgag acctcagaaa gaggcagtga gttccagtca cggacccagcgaccctacgg 1500 acagagcgga ggtggagaag gactcggggc acggcagcac ttccgtggattctgaggggt 1360 tcagcatccc agacacgggc agtcactgca gcagcgagta tgcggccagctccccagggg 1620 acaggggctc gcaggaacat gtggactctc aggagaaagc gcctgaaactgacgactctt 1680 tttcagatgt ggactgccat tcaaaccagg aagataccgg atgtaaatttcgagttttgc 1740 ctcagccaac taatctcgca accccaaaca caaagcgttt taaaaaagaagaaattcttt 1800 ccagttctga catttgtcaa aagttagtaa atactcagga catgtcagcctctcaggttg 1860 atgtagctgt gaaaattaat aagaaagttg tgcccctgga cttttctatgagttctttag 1920 ctaaacgaat aaagcagtta catcatgaag cacagcaaag tgaaggggaacagaattaca 1980 ggaagtttag ggcaaagatt tgtcctggag aaaatcaagc agccgaagatgaactaagaa 2040 aagagataag taaaacgatg tttgcagaaa tggaaatcat tggtcagtttaacctgggat 2100 ttataataac caaactgaat gaggatatct tcatagtgga ccagcatgccacggacgaga 2160 agtataactt cgagatgctg cagcagcaca ccgtgctcca ggggcagaggctcatagcac 2220 ctcagactct caacttaact gctgttaatg aagctgttct gatagaaaatctggaaatat 2280 ttagaaagaa tggctttgat tttgttatcg atgaaaatgc tccagtcactgaaagggcta 2340 aactgatttc cttgccaact agtaaaaact ggaccttcgg accccaggacgtcgatgaac 2400 tgatcttcat gctgagcgac agccctgggg tcatgtgccg gccttcccgagtcaagcaga 2460 tgtttgcctc cagagcctgc cggaagtcgg tgatgattgg gactgctcttaacacaagcg 2520 agatgaagaa actgatcacc cacatggggg agatggacca cccctggaactgtccccatg 2580 gaaggccaac catgagacac atcgccaacc tgggtgtcat ttctcagaactgaccgtagt 2640 cactgtatgg aataattggt tttatcgcag atttttatgt tttgaaagacagagtcttca 2700 ctaacctttt ttgttttaaa atgaaacctg ctacttaaaa aaaatacacatcacacccat 2760 ttaaaagtga tcttgagaac cttttcaaac c 2771 PMS1 (human)(SEQ ID NO: 11) MKQLPAATVR LLSSSQIITS VVSVVKELIE NSLDAGATSV 60DVKLENYGFD KIEVRDNGEG IKAVDAPVMA MKYYTSKINS HEDLENLTTY GFRGEALGSI 120CCIAEVLITT RTAADNFSTQ YVLDGSGHIL SQKPSHLGQG TTVTALRLFK NLPVRKQFYS 180TAKKCKDEIK KIQDLLMSFG ILKPDLRIVF VHNKAVIWQK SRVSDHKMAL MSVLGTAVMN 240NNESFQYHSE ESQIYLSGFL PKCDADHSFT SLSTPERSFI FINSRPVHQK DILKLIRHHY 300NLKCLKESTR LYPVFFLKID VPTADVDVNL TPDKSQVLLQ NKESVLIALE NLMTTCYGPL 360PSTNSYENNK TDVSAADIVL SKTAETDVLF NKVESSGKNY SNVDTSVIPF QNDMHNDESG 420KNTDDCLNHQ ISIGDFGYGH CSSEISNIDK NTKNAFQDIS MSNVSWENSQ TEYSKTCFIS 480SVKHTQSENG NKDHIDESGE NEEEAGLENS SEISADEWSR GNILKNSVGE NIEPVKILVP 540EKSLPCKVSN NNYPIPEQMN LNEDSCNKKS NVIDNKSGKV TAYDLLSNRV IKKPMSASAL 600FVQDHRPQFL IENPKTSLED ATLQIEELWK TLSEEEKLKY EEKATKDLER YNSQMKRAIE 660QESQMSLKDG RKKIKPTSAW NLAQKHKLKT SLSNQPKLDE LLQSQIEKRR SQNIKMVQIP 720FSMKNLKINF KKQNKVDLEE KDEPCLIHNL RFPDAWLMTS KTEVMLLNPY RVEEALLFKR 780LLENHKLPAE PLEKPIMLTE SLFNGSHYLD VLYKMTADDQ RYSGSTYLSD PRLTANGFKI 840KLIPGVSITE NYLEIEGMAN CLPFYGVADL KEILNAILNR NAKEVYECRP RKVISYLEGE 900AVRLSRQLPM YLSKEDIQDI IYRMKHQFGN EIKECVHGRP FFHHLTYLPE TT 932 PMS1(human) (SEQ ID NO: 12) ggcacgagtg gctgcttgcg gctagtggat ggtaattgcc 60tgcctcgcgc tagcagcaag ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc 120aacagttcga ctcctttcaa gttctcagat catcacttcg gtggtcagtg ttgtaaaaga 180gcttattgaa aactccttgg atgctggtgc cacaagcgta gatgttaaac tggagaacta 240tggatttgat aaaattgagg tgcgagataa cggggagggt atcaaggctg ttgatgcacc 300tgtaatggca atgaagtact acacctcaaa aataaatagt catgaagatc ttgaaaattt 360gacaacttac ggttttcgtg gagaagcctt ggggtcaatt tgttgtatag ctgaggtttt 420aattacaaca agaacggctg ctgataattt tagcacccag tatgttttag atggcagtgg 480ccacatactt tctcagaaac cttcacatct tggtcaaggt acaactgtaa ctgctttaag 540attatttaag aatctacctg taagaaagca gttttactca actgcaaaaa aatgtaaaga 600tgaaataaaa aagatccaag atctcctcat gagctttggt atccttaaac ctgacttaag 660gattgtcttt gtacataaca aggcagttat ttggcagaaa agcagagtat cagatcacaa 720gatggctctc atgtcagttc tggggactgc tgttatgaac aatatggaat cctttcagta 780ccactctgaa gaatctcaga tttatctcag tggatttctt ccaaagtgtg atgcagacca 840ctctttcact agtctttcaa caccagaaag aagtttcatc ttcataaaca gtcgaccagt 900acatcaaaaa gatatcttaa agttaatccg acatcattac aatctgaaat gcctaaagga 960atctactcgt ttgtatcctg ttttctttct gaaaatcgat gttcctacag ctgacgtcga 1020tgtaaattta acaccagata aaagccaagt attattacaa aataaggaat ctgttttaat 1080tgctcttgaa aatctgatga cgacttgtta tggaccatta cctagtacaa attcttatga 1140aaataataaa acagatgttt ccgcagctga catcgttctt agtaaaacag cagaaacaga 1200tgtgcttttt aataaagtgg aatcatctgg aaagaattat tcaaatgttg atacttcagt 1260cattccattc caaaatgata tgcataatga tgaatctgga aaaaacactg atgattgttt 1320aaatcaccag ataagtattg gtgactttgg ttatggtcat tgtagtagtg aaatttctaa 1380cattgataaa aacactaaga atgcatttca ggacatttca atgagtaatg tatcatggga 1440gaactctcag acggaatata gtaaaacttg ttttataagt tccgttaagc acacccagtc 1500agaaaatggc aataaagacc atatagatga gagtggggaa aatgaggaag aagcaggtct 1560tgaaaactct tcggaaattt ctgcagatga gtggagcagg ggaaatatac ttaaaaattc 1620agtgggagag aatattgaac ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa 1680agtaagtaat aataattatc caatccctga acaaatgaat cttaatgaag attcatgtaa 1740caaaaaatca aatgtaatag ataataaatc tggaaaagtt acagcttatg atttacttag 1800caatcgagta atcaagaaac ccatgtcagc aagtgctctt tttgttcaag atcatcgtcc 1860tcagtttctc atagaaaatc ctaagactag tttagaggat gcaacactac aaattgaaga 1920actgtggaag acattgagtg aagaggaaaa actgaaatat gaagagaagg ctactaaaga 1980cttggaacga tacaatagtc aaatgaagag agccattgaa caggagtcac aaatgtcact 2040aaaagatggc agaaaaaaga taaaacccac cagcgcatgg aatttggccc agaagcacaa 2100gttaaaaacc tcattatcta atcaaccaaa acttgatgaa ctccttcagt cccaaattga 2160aaaaagaagg agtcaaaata ttaaaatggt acagatcccc ttttctatga aaaacttaaa 2220aataaatttt aagaaacaaa acaaagttga cttagaagag aaggatgaac cttgcttgat 2280ccacaatctc aggtttcctg atgcatggct aatgacatcc aaaacagagg taatgttatt 2340aaatccatat agagtagaag aagccctgct atttaaaaga cttcttgaga atcataaact 2400tcctgcagag ccactggaaa agccaattat gttaacagag agtcttttta atggatctca 2460ttatttagac gttttatata aaatgacagc agatgaccaa agatacagtg gatcaactta 2520cctgtctgat cctcgtctta cagcgaatgg tttcaagata aaattgatac caggagtttc 2580aattactgaa aattacttgg aaatagaagg aatggctaat tgtctcccat tctatggagt 2640agcagattta aaagaaattc ttaatgctat attaaacaga aatgcaaagg aagtttatga 2700atgtagacct cgcaaagtga taagttattt agagggagaa gcagtgcgtc tatccagaca 2760attacccatg tacttatcaa aagaggacat ccaagacatt atctacagaa tgaagcacca 2820gtttggaaat gaaattaaag agtgtgttca tggtcgccca ttttttcatc atttaaccta 2880tcttccagaa actacatgat taaatatgtt taagaagatt agttaccatt gaaattggtt 2940ctgtcataaa acagcatgag tctggtttta aattatcttt gtattatgtg tcacatggtt 3000attttttaaa tgaggattca ctgacttgtt tttatattga aaaaagttcc acgtattgta 3060gaaaacgtaa ataaactaat aac 3063 MSH2 (human) (SEQ ID NO: 13) MAVQPKETLQLESAAEVGFV RFFQGMPEKP TTTVRLFDRG 60 DFYTAHGEDA LLAAREVFKT QGVIKYMGPAGAKNLQSVVL SKMNFESFVK DLLLVRQYRV 120 EVYKNRAGNK ASKENDWYLA YKASPGNLSQFEDILFGNND MSASIGVVGV KMSAVDGQRQ 180 VGVGYVDSIQ RKLGLCEFPD NDQFSNLEALLIQIGPKECV LPGGETAGDM GKLRQIIQRG 240 GILITERKKA DFSTKDIYQD LNRLLKGKKGEQMNSAVLPE MENQVAVSSL SAVIKFLELL 300 SDDSNFGQFE LTTFDFSQYM KLDIAAVRALNLFQGSVEDT TGSQSLAALL NKCKTPQGQR 360 LVNQWIKQPL MDKNRIEERL NLVEAFVEDAELRQTLQEDL LRRFPDLNRL AKKFQRQAAN 420 LQDCYRLYQG INQLPNVIQA LEKHEGKHQKLLLAVFVTPL TDLRSDFSKF QEMIETTLDM 480 DQVENHEFLV KPSFDPNLSE LREIMNDLEKKMQSTLISAA RDLGLDPGKQ IKLDSSAQFG 540 YYFRVTCKEE KVLRNNKNFS TVDIQKNGVKFTNSKLTSLN EEYTKNKTEY EEAQDAIVKE 600 IVNISSGYVE PMQTLNDVLA QLDAVVSFAHVSNGAPVPYV RPAILEKGQG RIILKASRMA 660 CVEVQDEIAF IPNDVYFEKD KQMFHIITGPNMGGKSTYIR QTGVIVLMAQ EGCFVPCESA 720 EVSIVDCILA RVGAGDSQLK GVSTFMAEMLETASILRSAT KDSLIIIDEL GRGTSTYDGF 780 GLAWAISEYI ATKIGAFCMF ATHFHELTALANQIPTVNNL HVTALTTEET LTMLYQVKKG 840 VCDQSFGIHV AELANFPKHV IECAKQKALELEEFQYIGES QGYDIMEPAA KKCYLEREQG 900 EKIIQEFLSK VKQMPFTEMS EENITIKLKQLKAEVIAKNN SFVNEIISRI KVTT 934 MSH2 (human cDNA) (SEQ ID NO: 14)ggcgggaaac agcttagtgg gtgtggggtc gcgcattttc 60 ttcaaccagg aggtgaggaggtttcgacat ggcggtgcag ccgaaggaga cgctgcagtt 120 ggagagcgcg gccgaggtcggcttcgtgcg cttctttcag ggcatgccgg agaagccgac 180 caccacagtg cgcctcttcgaccggggcga cttctatacg gcgcacggcg aggacgcgct 240 gctggccgcc cgggaggtgttcaagaccca gggggtgatc aagtacatgg ggccggcagg 300 agcaaagaat ctgcagagtgttgtgcttag taaaatgaat tttgaatctt ttgtaaaaga 360 tcttcttctg gttcgccagtatagagttga agtttataag aatagagctg gaaataaggc 420 atccaaggag aatgattggtatttggcata taaggcttct cctggcaatc tctctcagtt 480 tgaagacatt ctctttggtaacaatgatat gtcagcttcc attggtgttg tgggtgttaa 540 aatgtccgca gttgatggccagagacaggt tggagttggg tatgtggatt ccatacagag 600 gaaactagga ctgtgtgaattccctgataa tgatcagttc tccaatcttg aggctctcct 660 catccagatt ggaccaaaggaatgtgtttt acccggagga gagactgctg gagacatggg 720 gaaactgaga cagataattcaaagaggagg aattctgatc acagaaagaa aaaaagctga 780 cttttccaca aaagacatttatcaggacct caaccggttg ttgaaaggca aaaagggaga 840 gcagatgaat agtgctgtattgccagaaat ggagaatcag gttgcagttt catcactgtc 900 tgcggtaatc aagtttttagaactcttatc agatgattcc aactttggac agtttgaact 960 gactactttt gacttcagccagtatatgaa attggatatt gcagcagtca gagcccttaa 1020 cctttttcag ggttctgttgaagataccac tggctctcag tctctggctg ccttgctgaa 1080 taagtgtaaa acccctcaaggacaaagact tgttaaccag tggattaagc agcctctcat 1140 ggataagaac agaatagaggagagattgaa tttagtggaa gcttttgtag aagatgcaga 1200 attgaggcag actttacaagaagatttact tcgtcgattc ccagatctta accgacttgc 1260 caagaagttt caaagacaagcagcaaactt acaagattgt taccgactct atcagggtat 1320 aaatcaacta cctaatgttatacaggctct ggaaaaacat gaaggaaaac accagaaatt 1380 attgttggca gtttttgtgactcctcttac tgatcttcgt tctgacttct ccaagtttca 1440 ggaaatgata gaaacaactttagatatgga tcaggtggaa aaccatgaat tccttgtaaa 1500 accttcattt gatcctaatctcagtgaatt aagagaaata atgaatgact tggaaaagaa 1560 gatgcagtca acattaataagtgcagccag agatcttggc ttggaccctg gcaaacagat 1620 taaactggat tccagtgcacagtttggata ttactttcgt gtaacctgta aggaagaaaa 1680 agtccttcgt aacaataaaaactttagtac tgtagatatc cagaagaatg gtgttaaatt 1740 taccaacagc aaattgacttctttaaatga agagtatacc aaaaataaaa cagaatatga 1800 agaagcccag gatgccattgttaaagaaat tgtcaatatt tcttcaggct atgtagaacc 1860 aatgcagaca ctcaatgatgtgttagctca gctagatgct gttgtcagct ttgctcacgt 1920 gtcaaatgga gcacctgttccatatgtacg accagccatt ttggagaaag gacaaggaag 1980 aattatatta aaagcatccaggcatgcttg tgttgaagtt caagatgaaa ttgcatttat 2040 tcctaatgac gtatactttgaaaaagataa acagatgttc cacatcatta ctggccccaa 2100 tatgggaggt aaatcaacatatattcgaca aactggggtg atagtactca tggcccaaat 2160 tgggtgtttt gtgccatgtgagtcagcaga agtgtccatt gtggactgca tcttagcccg 2220 agtaggggct ggtgacagtcaattgaaagg agtctccacg ttcatggctg aaatgttgga 2280 aactgcttct atcctcaggtctgcaaccaa agattcatta ataatcatag atgaattggg 2340 aagaggaact tctacctacgatggatttgg gttagcatgg gctatatcag aatacattgc 2400 aacaaagatt ggtgctttttgcatgtttgc aacccatttt catgaactta ctgccttggc 2460 caatcagata ccaactgttaataatctaca tgtcacagca ctcaccactg aagagacctt 2520 aactatgctt tatcaggtgaagaaaggtgt ctgtgatcaa agttttggga ttcatgttgc 2580 agagcttgct aatttccctaagcatgtaat agagtgtgct aaacagaaag ccctggaact 2640 tgaggagttt cagtatattggagaatcgca aggatatgat atcatggaac cagcagcaaa 2700 gaagtgctat ctggaaagagagcaaggtga aaaaattatt caggagttcc tgtccaaggt 2760 gaaacaaatg ccctttactgaaatgtcaga agaaaacatc acaataaagt taaaacagct 2820 aaaagctgaa gtaatagcaaagaataatag ctttgtaaat gaaatcattt cacgaataaa 2880 agttactacg tgaaaaatcccagtaatgga atgaaggtaa tattgataag ctattgtctg 2940 taatagtttt atattgttttatattaaccc tttttccata gtgttaactg tcagtgccca 3000 tgggctatca acttaataagatatttagta atattttact ttgaggacat tttcaaagat 3060 ttttattttg aaaaatgagagctgtaactg aggactgttt gcaattgaca taggcaataa 3120 taagtgatgt gctgaattttataaataaaa tcatgtagtt tgtgg 3145 MLH1 (human) (SEQ ID NO: 15) MSFVAGVIRRLDETVVNRIA AGEVIQRPAN AIKEMIENCL 60 DAKSTSIQVI VKEGGLKLIQ IQDNGTGIRKEDLDIVCERF TTSKLQSFED LASISTYGFR 120 GEALASISHV AHVTITTKTA DGKCAYRASYSDGKLKAPPK PCAGNQGTQI TVEDLFYNIA 180 TRRKALKNPS EEYGKILEVV GRYSVHNAGISFSVKKQGET VADVRTLPNA STVONIRSIF 240 GNAVSRELIE IGCEDKTLAE KMNGYISNANYSVKKCIFLL EINHRLVEST SLRKAIETVY 300 AAYLPKNTHP FLYLSLEISP QNVDVNVHPTKHEVHFLHEE SILERVQQHI ESKLLGSNSS 360 RMYFTQTLLP GLAGPSGEMV KSTTSLTSSSTSGSSDKVYA HQMVRTDSRE QKLDAELQPL 420 SKPLSSQPQA IVTEDKTDIS SGRARQQDEEMLELPAPAEV AAKNQSLEGD TTKGTSEMSE 480 KRGPTSSNPR KRHREDSDVE MVEDDSRKEMTAACTPRRRI INLTSVLSLQ EEINEQGHEV 540 LREMLHNHSF VGCVNPQWAL AQHQTKLYLLNTTKLSEELF YQILIYDFAN FGVLRLSEPA 600 PLFDLAMLAL DSPESGWTEE DGPKEGLAEYIVEFLKKKAE MLADYFSLEI DEEGNLIGLP 660 LLIDNYVPPL EGLPIFILRL ATEVNWDEEKECFESLSKEC AMFYSIRKOY ISEESTLSGQ 720 QSEVPGSIPN SWKWTVEHIV YKALRSHILPPKHFTEDGNI LQLANLPDLY KVFERC 756 MLHI (human) (SEQ ID NO: 16) cttggctcttctggcgccaa aatgtcgttc gtggcagggg 60 ttattcggcg gctggacgag acagtggtgaaccgcatcgc ggcgggggaa gttatccagc 120 ggccagctaa tgctatcaaa gagatgattgagaactgttt agatgcaaaa tccacaagta 180 ttcaagtgat tgttaaagag ggaggcctgaagttgattca gatccaagac aatggcaccg 240 ggatcaggaa agaagatctg gatattgtatgtgaaaggtt cactactagt aaactgcagt 300 cctttgagga tttagccagt atttctacctatggctttcg aggtgaggct ttggccagca 360 taagccatgt ggctcatgtt actattacaacgaaaacagc tgatggaaag tgtgcataca 420 gagcaagtta ctcagatgga aaactgaaagcccctcctaa accatgtgct ggcaatcaag 480 ggacccagat cacggtggag gaccttttttacaacatagc cacgaggaga aaagctttaa 540 aaaatccaag tgaagaatat gggaaaattttggaagttgt tggcaggtat tcagtacaca 600 atgcaggcat tagtttctca gttaaaaaacaaggagagac agtagctgat gttaggacac 660 tacccaatgc ctcaaccgtg gacaatattcgctccatctt tggaaatgct gttagtcgag 720 aactgataga aattggatgt gaggataaaaccctagcctt caaaatgaat ggttacatat 780 ccaatgcaaa ctactcagtg aagaagtgcatcttcttact cttcatcaac catcgtctgg 840 tagaatcaac ttccttgaga aaagccatagaaacagtgta tgcagcctat ttgcccaaaa 900 acacacaccc attcctgtac ctcagtttagaaatcagtcc ccagaatgtg gatgttaatg 960 tgcaccccac aaagcatgaa gttcacttcctgcacgagga gagcatcctg gagcgggtgc 1020 agcagcacat cgagagcaag ctcctgggctccaattcctc caggatgtac ttcacccaga 1080 ctttgctacc aggacttgct ggcccctctggggagatggt taaatccaca acaagtctga 1140 cctcgtcttc tacttctgga agtagtgataaggtctatgc ccaccagatg gttcgtacag 1200 attcccggga acagaagctt gatgcatttctgcagcctct gagcaaaccc ctgtccagtc 1260 agccccaggc cattgtcaca gaggataagacagatatttc tagtggcagg gctaggcagc 1320 aagatgagga gatgcttgaa ctcccagcccctgctgaagt ggctgccaaa aatcagagct 1380 tggaggggga tacaacaaag gggacttcagaaatgtcaga gaagagagga cctacttcca 1440 gcaaccccag aaagagacat cgggaagattctgatgtgga aatggtggaa gatgattccc 1500 gaaaggaaat gactgcagct tgtaccccccggagaaggat cattaacctc actagtgttt 1560 tgagtctcca ggaagaaatt aatgagcagggacatgaggt tctccgggag atgttgcata 1620 accactcctt cgtgggctgt gtgaatcctcagtgggcctt ggcacagcat caaaccaagt 1680 tataccttct caacaccacc aagcttagtgaagaactgtt ctaccagata ctcatttatg 1740 attttgccaa ttttggtgtt ctcaggttatcggagccagc accgctcttt gaccttgcca 1800 tgcttgcctt agatagtcca gagagtggctggacagagga agatggtccc aaagaaggac 1860 ttgctgaata cattgttgag tttctgaagaagaaggctga gatgcttgca gactatttct 1920 ctttggaaat tgatgaggaa gggaacctgattggattacc ccttctgatt gacaactatg 1980 tgcccccttt ggagggactg cctatcttcattcttcgact agccactgag gtgaattggg 2040 acgaagaaaa ggaatgtttt gaaagcctcagtaaagaatg cgctatgttc tattccatcc 2100 ggaagcagta catatctgag gagtcgaccctctcaggcca gcagagtgaa gtgcctggct 2160 ccattccaaa ctcctggaag tggactgtggaacacattgt ctataaagcc ttgcgctcac 2220 acattctgcc tcctaaacat ttcacagaagatggaaatat cctgcagctt gctaacctgc 2280 ctgatctata caaagtcttt gagaggtgttaaatatggtt atttatgcac tgtgggatgt 2340 gttcttcttt ctctgtattc cgatacaaagtgttgtatca aagtgtgata tacaaagtgt 2400 accaacataa gtgttggtag cacttaagacttatacttgc cttctgatag tattccttta 2460 tacacagtgg attgattata aataaatagatgtgtcttaa cata 2484 hPMS2-134 (human) (SEQ ID NO: 17) MERAESSSTEPAKAIKPIDR KSVHQICSGQ VVLSLSTAVK 60 ELVENSLDAG ATNIDLKLKD YGVDLIEVSDNGCGVEEENF EGLTLKHHTS KIQEFADLTQ 120 VETFGFRGEA LSSLCALSDV TISTCHASAKVGT 133 hPMS2-134 (human cDNA) (SEQ ID NO: 18) cgaggcggat cgggtgttgcatccatggag cgagctgaga 60 gctcgagtac agaacctgct aaggccatca aacctattgatcggaagtca gtccatcaga 120 tttgctctgg gcaggtggta ctgagtctaa gcactgcggtaaaggagtta gtagaaaaca 180 gtctggatgc tggtgccact aatattgatc taaagcttaaggactatgga gtggatctta 240 ttgaagtttc agacaatgga tgtggggtag aagaagaaaacttcgaaggc ttaactctga 300 aacatcacac atctaagatt caagagtttg ccgacctaactcaggttgaa acttttggct 360 ttcgggggga agctctgagc tcactttgtg cactgagcgatgtcaccatt tctacctgcc 420 acgcatcggc gaaggttgga acttga 426

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples, which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLES Example 1 hPMS2-134 Encodes a Dominant Negative Mismatch RepairProtein

A profound defect in MMR was found in the normal cells of two HNPCCpatients. That this defect was operative in vivo was demonstrated by thewidespread presence of microsatellite instability in non neoplasticcells of such patients. One of the two patients had a germ linetruncating mutation of the hPMS2 gene at codon 134 (the hPMS2 134mutation), while the other patient had a small germ line deletion withinthe hMLH1 gene. Leach et al., Cell, 1993, 75, 1215 1225. These datacontradicted the two hit model generally believed to explain thebiochemical and biological features of HNPCC patients. The basis forthis MMR deficiency in the normal cells of these patients was unclear,and several potential explanations were offered. For example, it waspossible that the second allele of the relevant MMR gene was inactivatedin the germ line of these patients through an undiscovered mechanism, orthat unknown mutations of other genes involved in the MMR process werepresent that cooperated with the known germ line mutation. It is clearfrom knock out experiments in mice that MMR deficiency is compatiblewith normal growth and development, supporting these possibilities.Edelmann et al., Cell, 1996, 85, 1125 1134. Alternatively, it waspossible that the mutant alleles exerted a dominant-negative effect,resulting in MMR deficiency even in the presence of the wild type alleleof the corresponding MMR gene and all other genes involved in the MMRprocess. To distinguish between these possibilities, the truncatedpolypeptide encoded by the hPMS2 134 mutation was expressed in an MMRproficient cell line its affect on MMR activity was analyzed. Theresults showed that this mutant could indeed exert a dominant-negativeeffect, resulting in biochemical and genetic manifestations of MMRdeficiency. One embodiment of the present invention is demonstrated inTable 1, where a Syrian hamster fibroblast cell line (TK) wastransfected with an expression vector containing the hPMS2-134(TK-PMS2-134) or the empty expression vector (TKvect), which alsocontains the NEO gene as a selectable marker. TK-PMS2-134 cells weredetermined to be stably expressing the gene via western blot analysis(data not shown). Nuclear lysates from hPMS2-134 and control cells weremeasured for the ability to correct mismatched DNA substrates. As shownin Table 1, TK-PMS2-134 cells had a dramatic decrease in repair activitywhile TKvect control cells were able to repair mismatched DNA duplexesat a rate of ˜4.0 fmol/15 minutes (p<0.01). TABLE 1 Relative endogenousMMR activity of MMR-proficient cells expressing an ectopically expressedmorphogene or an empty expression vector 5′ DNA Repair activity of G/Tmismatch Cell Lines (fmol/15 minutes) TKvect 1 3.5 2 2.9 3 5.5TK-PMS2-134 1 0 2 0 3 0.5These data show that the expression of the TK-PMS2-134 results insuppressed MMR of a host organism and allows for an enhanced mutationrate of genetic loci with each mitosis.

Example 2 hPMS2-134 Can Alter Genes In Vivo

An example of the ability to alter mismatch repair comes fromexperiments using manipulation of mismatch repair TK cells (describedabove) that expressed the TK-hPMS2-134 mutant were used by transfectionof the mammalian expression construct containing a defectiveβ-galactosidase gene (referred to as pCAR-OF) which was transfected intoTK-hPMS2-134 or TKvect cells as described above. The pCAR OF vectorconsists of a β-galactosidase gene containing a 29-basepair poly-CAtract inserted at the 5′ end of its coding region, which causes thewild-type reading frame to shift out-of-frame. This chimeric gene iscloned into the pCEP4, which contains the constitutively activecytomegalovirus (CMV) promoter upstream of the cloning site and alsocontains the hygromycin-resistance gene that allows for selection ofcells containing this vector. The pCAR-OF reporter cannot generateβ-galactosidase activity unless a frame-restoring mutation (i.e.,insertion or deletion) arises following transfection into a host.Another reporter vector called pCAR-IF contains a β-galactosidase inwhich a 27-bp poly-CA repeat was cloned into the same site as thepCAR-OF gene, but it is biologically active because the removal of asingle repeat restores the open reading frame and produces a functionalchimeric β-galactosidase polypeptide (not shown). In these experiments,TK-hPMS2-134 and TKvect cells were transfected with the pCAR-OF reportervector and selected for 17 days in neomycin plus hygromycin selectionmedium. After the 17 days, resistant colonies were stained forβ-galactosidase production to determine the number of clones containinga genetically altered β-galactosidase gene. All conditions produced arelatively equal number of neomycin/hygromycin resistant cells, however,only the cells expressing the TK-hPMS2-134 contained a subset of clonesthat were positive for β-galactosidase activity. Representative resultsare shown in Table 2, which shows the data from these experiments wherecell colonies were stained in situ for β-galactosidase activity andscored for activity. Cells were scored positive if the colonies turnedblue in the presence of X-gal substrate and scored negative if coloniesremained white. Analysis of triplicate experiments showed that asignificant increase in the number of functional β-galactosidasepositive cells was found in the TK-hPMS2-134 cultures, while noβ-galactosidase positive cells were seen in the control TKvect cells.TABLE 2 Number of TKmorph and TKvect cells containing functionalβ-galactosidase activity % Clones with Cells White Colonies BlueColonies altered B-gal Tkvect 65 +/− 9  0  0/65 = 0% TK-PMS2-134 40 +/−12 28 +/− 4 28/68 = 41%TK-PMS2-134/pCAR-OF clones that were pooled and expanded also showed anumber of cells that contained a functional β-galactosidase gene. Noβ-galactosidase positive cells were observed in TKvect cells transfectedwith the pCAR-OF vector. These data are shown in FIG. 1 where the darkstaining in panel B represent β-galactosidase positive cells present inthe TK-PMS2-134/pCAR-OF cultures while none are found in the TKvectcells grown under similar conditions (panel A). These data demonstratethe ability of the mutant mismatch repair gene, hPMS2-134, to generategene alterations in vivo, which allows for the rapid screening of cloneswith altered polypeptides exhibiting new biochemical features.

To confirm that alterations within the nucleotide sequences of theβ-galactosidase gene was indeed responsible for the in vivoβ-galactosidase activity present in TK-hPMS2-134 clones, RNA wasisolated from TK-hPMS2-134/pCAR-OF and TKvect/pCAR-OF and theβ-galactosidase mRNA primary structure was examined by reversetranscriptase polymerase chain reaction (RT-PCR) amplification andsequencing. Sequence analysis of α-galactosidase message from TKvectcells found no structural alterations in the input gene sequence.Analysis of the β-galactosidase message from TK-hPMS-134 cells foundseveral changes within the coding sequences of the gene. These sequencealterations included insertion and deletions of the poly CA tract in theamino terminus as expected. Other alterations included insertions ofsequences outside of the polyCA repeat as well as a series of singlebase alterations contained throughout the length of the gene.

A summary of the genetic alterations are given in FIG. 2 where aschematic representation of the β-galactosidase gene is shown with theregions and types of genetic alterations depicted below.

Plasmids. The full length wild type hPMS2 cDNA was obtained from a humanHela cDNA library as described in Strand et al., Nature, 1993, 365, 274276, which is incorporated herein by reference in its entirety. An hPMS2cDNA containing a termination codon at amino acid 134 was obtained viaRT PCR from the patient in which the mutation was discovered. Nicolaideset al., Mol. Cell. Biol., 1998, 18, 1635-1641, which is incorporatedherein by reference in its entirety. The cDNA fragments were cloned intothe BamHI site into the pSG5 vector, which contains an SV40 promoterfollowed by an SV40 polyadenylation signal. Nicolaides et al., Genomics,1995, 29, 329 334, which is incorporated herein by reference in itsentirety. The pCAR reporter vectors described in FIG. 1 were constructedas described in Palombo et al., Nature, 1994, 36, 417, which isincorporated herein by reference in its entirety.

β-galactosidase assay. Seventeen days following transfection with pCAR,β-galactosidase assays were performed using 20 μg of protein in 45 mM 2mercaptoethanol, 1 mM MgCl₂, 0.1 M NaPO₄ and 0.6 mg/ml Chlorophenol redβ-D galatopyranoside (CPRG, Boehringer Mannheim). Reactions wereincubated for 1 hour, terminated by the addition of 0.5 M Na₂CO₃, andanalyzed by spectrophotometry at 576 nm. Nicolaides et al., Mol. Cell.Biol., 1998, 18, 1635-1641. For in situ β-galactosidase staining, cellswere fixed in 1% glutaraldehyde in PBS and incubated in 0.15 M NaCl, 1mM MgCl₂, 3.3 mM K₄Fe(CN)₆, 3.3 mM K₃Fe(CN)₆, 0.2% X Gal for 2 hours at37° C.

Example 3 hPMS2-134 Causes a Defect in MMR Activity

The differences in β-galactosidase activity between PMS2 WT and PMS2 134transfected cells can be due to the PMS2 134 protein disturbing MMRactivity resulting in a higher frequency of mutation within the pCAR OFreporter and re establishing the ORF. To directly test whether MMR wasaltered, a biochemical assay for MMR with the individual clonesdescribed in Example 1 was employed. Nuclear extracts were prepared fromthe clones and incubated with heteroduplex substrates containing eithera /CA\ insertion deletion or a G/T mismatch under conditions describedpreviously. The /CA\ and G/T heteroduplexes were used to test repairfrom the 3′ and 5′ directions, respectively. There was a dramaticdifference between the PMS2-134 expressing clones and the other clonesin these assays (Table 3). TABLE 3 MMR activity of nuclear extracts fromSH clones or pooled culturesa Amt of repaired substrate (fmol/15 min)Cell line 3′/CA\3′G/T 5′ G/T 3′/CTG\ 5′/CTG\ SH clonesb PMS2-NOT Clone A10.2 3.5 Clone B 12.7 2.9 Clone C 13.5 5.5 PMS2-WT Clone A 2.8 2.2 CloneB 5.7 4.8 Clone c 4.7 2.9 PMS2-134 Clone A 2.5 0.0 Clone B 2.4 0.0 CloneC 5.0 0.5 Pooled cultures PMS2-NOT 2.07 ± 0.09 2.37 ± 0.37 3.45 ± 1.352.77 ± 1.37 PMS2-WT 1.65 ± 0.94 1.86 ± 0.57 1.13 ± 0.23 1.23 ± 0.65PMS2-134 0.14 ± 0.2  0.0 ± 0.0 1.31 ± 0.66  0.0 ± 0.0aThe extracts were tested for MMR activity with 24 fmol of heteroduplex.bThese data represent similar results derived from more than fiveindependent experiments.While all clones repaired substrates from the 3′ direction (/CA\heteroduplex), cells expressing the PMS2 134 polypeptide had very little5′ repair activity. A similar directional defect in mismatch repair wasevident with pooled clones resulting from PMS2 134 transfection, or whenthe heteroduplex contained a 2 4 base pair loop, examples of which areshown in Table 3. A small decrease in MMR activity was observed in the3′/CA\ PMS2-WT repair assays, perhaps a result of interference in thebiochemical assays by over-expression of the PMS2 protein. Nosignificant activity was caused by PMS2-WT in the in situβ-galactosidase assays, a result more likely to reflect the in vivocondition.

Biochemical assays for mismatch repair. MMR activity in nuclear extractswas performed as described, using 24 fmol of substrate, in Bronner etal., Nature, 1994, 368, 258 261 and Nicolaides et al., Mol. Cell. Biol.,1998, 18, 1635-1641, each of which is incorporated herein by referencein its entirety. Complementation assays were done by adding ˜100 ng ofpurified MutL″ or MutS″ components to 100 μg of nuclear extract,adjusting the final KCl concentration to 100 mM. Bevins, Ciba Found.Symp., 1994, 186, 250-69 and Alderson et al., Res. Microbiol., 1993,144, 665-72. The substrates used in these experiments contain a strandbreak 181 nucleotides 5′ or 125 nucleotides 3′ to the mismatch. Valuesrepresent experiments performed at least in duplicate.

Example 4 C-Terminus of hPMS2 Mediates Interaction Between hPMS2 andhMLH1

To elucidate the mechanism by which hPMS2 134 affected MMR, theinteraction between hPMS2 and hMLH1 was analyzed. Previous studies haveshown that these two proteins dimerize to form a functionally activecomplex. Bronner et al., Nature, 1994, 368, 258 261. Proteins weresynthesized in vitro using reticulocyte lysates, employing RNA generatedfrom cloned templates. The full length hMLH1 and hPMS2 proteins bound toeach other and were co precipitated with antibodies to either protein,as expected (data not shown). To determine the domain of hPMS2 thatbound to hMLH1, the amino terminus (codons 1-134), containing the mosthighly conserved domain among mutL proteins (Su et al., J. Biol. Chem.,1988, 263, 6829 6835 and Edelmann et al., Cell, 1996, 85, 1125 1134),and the carboxyl terminus (codons 135-862) were separately cloned andproteins produced in vitro in coupled transcription translationreactions. FIGS. 3A, 3B, and 3C show a representativeimmunoprecipitation of in vitro-translated hPMS2 and hMLH1 proteins.FIG. 3A shows labeled (indicated by an asterisk) or unlabelled proteinsincubated with an antibody to the C-terminus of hPMS2 in lanes 1 to 3and to hMLH1 in lanes 4 to 6. Lane 7 contains a nonprogrammedreticulocyte lysate. PMS2-135 contains codons 135 to 862 of hPMS2. Themajor translation products of hPMS2 and hMLH1 are indicated. FIG. 3Bshows labeled hPMS2-134 (containing codons 1-134 of hPMS2) incubated inthe presence or absence of unlabelled hMLH1 plus an antibody to hMLH1(lanes 1 and 2, respectively). Lane 3 contains lysate from anonprogrammed reticulolysate. FIG. 3C shows labeled proteins incubatedwith an antibody to the N terminus of hPMS2. Lane 6 contains anonprogrammed reticulocyte lysate. In both panels A and B,autoradiographs of immunoprecipitated products are shown. When a 35Slabelled, full-length hMLH1 protein (FIG. 3A, lane 5) was mixed with theunlabelled carboxyl terminal hPMS2 polypeptide, a monoclonal antibody(mAb) to the carboxyl terminus of hPMS2 efficiently immunoprecipitatedthe labeled hMLH1 protein (lane 1). No hMLH1 protein was precipitated inthe absence of hPMS2 (lane 2). Conversely, when the 35S labelledcarboxyl terminus of hPMS2 (lane 3) was incubated with unlabelled, fulllength hMLH1 protein, an anti hMLH1 mAb precipitated the hPMS2polypeptide (lane 4). In the absence of the unlabelled hMLH1 protein, nohPMS2 protein was precipitated by this mAb (lane 6). The same antibodyfailed to immunoprecipitate the amino terminus of hPMS2 (amino acids 1134) when mixed with unlabelled MLH1 protein (FIG. 3B, lane 1). Thisfinding was corroborated by the converse experiment in whichradiolabelled hPMS2-134 (FIG. 3C, lane 1) was unable to coprecipitateradiolabelled MLH1 when precipitations were done using an N terminalhPMS2 antibody (FIG. 3C, lane 2) while this antibody was shown to beable to coprecipitate MLH1 when mixed with wild type hPMS2 (FIG. 3C,lane 4).

The initial steps of MMR are dependent on two protein complexes, calledMutS″ and MutL″. Drummond et al., Science, 1995, 268, 1909 1912. As theamino terminus of hPMS2 did not mediate binding of hPMS2 to hMLH1, itwas of interest to determine whether it might instead mediate theinteraction between the MutL″ complex (comprised of hMLH1 and hPMS2) andthe MutS″ complex (comprised of MSH2 and GTBP). Because previous studieshave demonstrated that MSH2 and the MutL″ components do not associate insolution, direct hPMS2-134:MutS″ interaction was unable to be assayed. Adifferent approach was used to address this issue, and attempted tocomplement nuclear extracts from the various SH cell lines with MutS″ orMutL″. If the truncated protein present in the PMS2-134 expressing SHcells was binding to MutS″ and lowering its effective concentration inthe extract, then adding intact MutS″ should rescue the MMR defect insuch extracts. FIG. 4 shows complementation of MMR activity intransduced SH cells. Lysates from pooled clones stably transduced withPMS2-NOT, PMS2-WT, or PMS2-134 were complemented with purified MutS″ orMutL″ MMR components by using the 5′ G/T heteroduplex substrate. Thevalues are presented as the percentage of repair activity in each casecompared to that in lysates complemented with both purified MutL″ andMutS″ components to normalize for repair efficiency in the differentlysate backgrounds. The values shown represent the average of at leastthree different determinations. Purified MutS″ added to such extractshad no effect (FIG. 4). In contrast, addition of intact MutL″ to theextract completely restored directional repair to the extracts fromPMS2-134 cells (FIG. 4).

The results described above lead to several conclusions. First,expression of the amino terminus of hPMS2 results in an increase inmicrosatellite instability, consistent with a replication error (RER)phenotype. That this elevated microsatellite instability is due to MMRdeficiency was proven by evaluation of extracts from stably transducedcells. Interestingly, the expression of PMS2-134 resulted in a polardefect in MMR, which was only observed using heteroduplexes designed totest repair from the 5′ direction (no significant defect in repair fromthe 3′ direction was observed in the same extracts). Interestingly,cells deficient in hMLH1 also have a polar defect in MMR, but in thiscase preferentially affecting repair from the 3′ direction. Huttner etal., Pediatr. Res., 1999, 45, 785-94. It is known from previous studiesin both prokaryotes and eukaryotes that the separate enzymaticcomponents mediate repair from the two different directions. Our resultsindicate a model in which 5′ repair is primarily dependent on hPMS2while 3′ repair is primarily dependent on hMLH1. It is easy to envisionhow the dimeric complex between PMS2 and MLH1 might set up thisdirectionality. The combined results also demonstrate that a defect indirectional MMR is sufficient to produce a RER+ phenotype.

The dominant-negative function of the PMS2-134 polypeptide can resultfrom its binding to MLH1 and consequent inhibition of MutL″ function.This is based in part on the fact that the most highly conserved domainof the PMS2 gene is located in its amino terminus, and the only knownbiochemical partner for PMS2 is MLH1. The binding studies revealed,however, that the carboxyl terminus of PMS2, rather than the highlyconserved amino terminus, actually mediated binding to MLH1. This resultis consistent with those recently obtained in S. cerevisciae, in whichthe MLH1 interacting domain of PMS1 (the yeast homolog of human PMS2)was localized to its carboxyl terminus. Leach et al., Cell, 1993, 75,1215 1225. The add back experiments additionally showed that thehPMS2-134 mutant was not likely to mediate an interaction with the MutS″complex (FIG. 4). The hPMS2-134 polypeptide does not inhibit the initialsteps in MMR, but rather interacts with and inhibits a downstreamcomponent of the pathway, perhaps a nuclease required for repair fromthe 5′ direction.

The demonstration that the hPMS2-134 mutation can confer adominant-negative MMR defect to transfected cells helps to explain thephenotype of the kindred in which this mutant was discovered. Threeindividuals from this kindred were found to carry the mutation, a fatherand his two children. Both children exhibited microsatellite instabilityin their normal tissues and both developed tumors at an early age, whilethe father had no evidence of microsatellite instability in his normalcells and was completely healthy at age 35. The only difference known tous with respect to the MMR genes in this family is that the father'smutant allele was expressed at lower levels than the wild type allele asassessed by sequencing of RT PCR products of RNA from lymphocytes. Thechildren expressed both alleles at approximately equal levels. Thedominant negative attribute of the hPMS2-134 mutant may only be manifestwhen it is present at sufficient concentrations (at least equimolar)thus, explaining the absence of MMR deficiency in the father. The reasonfor the differential expression of the hPMS2-134 allele in this kindredis not clear, though imprinting is a possibility. Ascertainment ofadditional, larger kindreds with such mutations will facilitate theinvestigation of this issue.

Western blots. Equal number of cells were lysed directly in lysis buffer(60 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1 M 2 mercaptoethanol,0.001% bromophenol blue) and boiled for 5 minutes. Lysate proteins wereseparated by electrophoresis on 4 12% Tris glycine gels (for analysis offull length hPMS2) or 4 20% Tris glycine gels (for analysis ofhPMS2-134). Gels were electroblotted onto Immobilon P (Millipore) in 48mM Tris base, 40 mM glycine, 0.0375% SDS, 20% methanol and blockedovernight at 4° C. in Tris buffered saline plus 0.05% Tween 20 and 5%condensed milk. Filters were probed with a polyclonal antibody generatedagainst residues 2-20 of hPMS2 (Santa Cruz Biotechnology, Inc.) and ahorseradish peroxidase conjugated goat anti rabbit secondary antibody,using chemiluminescence for detection (Pierce).

In vitro translation. Linear DNA fragments containing hPMS2 and hMLH1cDNA sequences were prepared by PCR, incorporating sequences for invitro transcription and translation in the sense primer. A full lengthhMLH1 fragment was prepared using the sense primer 5′ggatcctaatacgactcactatagggaga ccaccatgtcgttcgtggcaggg 3′ (SEQ ID NO:1)(codons 1-6) and the antisense primer 5′ taagtcttaagtgctaccaac 3′ (SEQID NO:2) (located in the 3′ untranslated region, nt 2411 2433), using awild type hMLH1 cDNA clone as template. A full length hPMS2 fragment wasprepared with the sense primer 5′ ggatcctaatacgactcactatagggagaccaccatggaacaattgcctgcgg 3′ (SEQ ID NO:3) (codons 1-6) and theantisense primer 5′ aggttagtgaagactctgtc 3′ (SEQ ID NO:4) (located in 3′untranslated region, nt 2670 2690) using a cloned hPMS2 cDNA astemplate. A fragment encoding the amino terminal 134 amino acids ofhPMS2 was prepared using the same sense primer and the antisense primer5′ agtcgagttccaaccttcg 3′ (SEQ ID NO:5). A fragment containing codons135-862 of hPMS135 was generated using the sense primer 5′ggatcctaatacgactcactatagggagaccaccatgatgtttgatcacaatgg 3′ (SEQ ID NO:6)(codons 135-141) and the same antisense primer as that used for the fulllength hPMS2 protein. These fragments were used to produce proteins viathe coupled transcription translation system (Promega). The reactionswere supplemented with ³⁵S labelled methionine or unlabelled methionine,as indicated in the text. The PMS135 and hMLH1 proteins could not besimultaneously radiolabelled and immunoprecipitated because of theirsimilar molecular weights precluded resolution. Lower molecular weightbands are presumed to be degradation products and/or polypeptidestranslated from alternative internal methionines.

Immunoprecipitation. Immunoprecipitations were performed on in vitrotranslated proteins by mixing the translation reactions with 1 μg of theMLH1 specific monoclonal antibody (mAB) MLH14 (Oncogene Science, Inc.),a polyclonal antibody generated to codons 2-20 of hPMS2 described above,or a polyclonal antibody generated to codons 843-862 of hPMS2 (SantaCruz Biotechnology, Inc.) in 400:1 of EBC buffer (50 mM Tris, pH 7.5,0.1 M NaCl, 0.5% NP40). After incubation for 1 hour at 4° C., protein Asepharose (Sigma) was added to a final concentration of 10% andreactions were incubated at 4° C. for 1 hour. Proteins bound to proteinA were washed five times in EBC and separated by electrophoresis on 420% Tris glycine gels, which were then dried and autoradiographed.

Example 5 Syrian Hamster Tk-ts-13 Cells Produce a Novel Anti-MicrobialPolypeptide Can Suppress the Growth of Bacillus subtilis

The feasibility of creating microbial-resistant mammalian cells isdemonstrated as follows. Syrian Hamster TK fibroblasts were transfectedwith a mammalian expression vector containing a novel anti-microbialpolypeptide called mlg1 or the empty expression vector called psg. Whencells expressing the mlg polypeptide (referred to as TK-mlg1) were grownin the presence of Bacillus subtilis, these cells were able to suppressthe growth of the microbes and allow the TK host to remain viable incontrast to TK cells transfected with the empty vector (TK=psg), whichall died from the toxic effects that Bacillus subtilis has on mammaliancells. FIG. 5 shows a photograph of TK-mlg1 and TK=psg cultures grown inthe presence of Bacillus for 4 days. Syrian hamster Tk-ts13 cells weretransfected with a eukaryotic expression vector that produces a novelantimicrobial polypeptide referred to as mlg1 (Panel A) or theexpression vector lacking an inserted cDNA for expression (TK=psg, PanelB). Cells were plated at a density of 5×10⁵ cells/ml in a 10 cm falconpyrogenic-free petri dish in growth medium for 24 hours and theninoculated with 10:1 of an exponentially growing culture of Bacillussubtilis. Cultures were then incubated for 4 days at which time Bacilligrow and begin to lyse the Tk-ts13 parental culture as shown in panel B(indicated by arrows), while cells expressing the anti-microbialmlg1polypeptide (Panel A) remain viable in the presence of Bacillus(small granular structures present in panels A and B). These datademonstrate the feasibility of cells to survive in the presence ofBacillus contamination when they produce an anti-microbial agent. Thesedata show that antimicrobial producing mammalian cells are capable ofgrowing and surviving in the presence of toxic microbes.

Cell lines and transfection. Syrian Hamster fibroblast Tk ts13 cellswere obtained from ATCC and cultured as described. Modrich, Science,1994, 266, 1959 1960. Stably transfected cell lines expressing hPMS2were created by cotransfection of the PMS2 expression vectors and thepLHL4 plasmid encoding the hygromycin resistance gene at a ratio of 3:1(pCAR:pLHL4) and selected with hygromycin. Stably transfected cell linescontaining pCAR reporters were generated by co transfection of pCARvectors together with either pNTK plasmid encoding the neomycinresistance plasmid or with pLHL4. All transfections were performed usingcalcium phosphate as previously described in Modrich, Science, 1994,266, 1959 1960, which is incorporated herein by reference in itsentirety.

Example 6 TK-hPMS2-134 Cells Can Suppress the Growth of Escherichia coliIn Vitro

While TK-hPMS2-134 TK-ts13 cells have been previously shown to becapable of altering genes in vivo (refer to Table 2 and FIG. 1), theability to generate “naturally microbial-resistant” clones has not beenreported in the literature. To generate microbial-resistant TK cells,TK-ts13 cells constitutively expressing the a dominant-negative mismatchrepair gene, TK-hPMS2-134 or the empty vector (TKvect) that have been inculture for >3 months (˜60 passages) were seeded at 5×10⁵ cells/ml inDulbeicco's Modified Eagles Medium (DMEM) plus 10% fetal bovine serum(FBS) and plated into 10 cm dishes (Falcon) in duplicate. These cellswere grown overnight at 37° C. in 5% CO₂ to allow cells to adhere to theplastic. The next day, TK cultures were inoculated with 10:1 of anexponentially growing culture of Escherichia coli. Cultures were thengrown at 37° C. in 5% CO₂ and observed on day 7 and 14 formicrobial-resistant cell clones; these cells appear as clones of cellssurrounded by “cleared” areas on the plate. At day 7, all cells in thecontrol transfected TKvect culture were dead, while a subset of cellswere viable in the TK-hPMS2-134 transfected cultures. At day 14, therewere no clones in the control transfected TKvect cultures, while therewere 34 and 40 Escherichia coli-resistant colonies formed in theTK-hPMS2-134 transfected cultures. Growing clones from each dish werethen pooled as individual cultures and grown to confluence. Thesecultures were named TK-hPMS2-134 (R1) and TK-hPMS2-134 (R2). Cultureswere cured of Escherichia coli by the addition of 1 mg/ml G418, in whichthe TK-hPMS2-134 cells are resistant due to expression of theneomycin-resistance gene contained on the mammalian expression vectorused to generate the cells.

TKvect, TK-hPMS2-134 (1) and TK-hPMS2-134 (R2) cells were plated at5×10⁵ cell/ml in 10 mls and plated into 10 cm dishes in duplicate. Thenext day, 10:1 of a logarithmic stage Escherichia coli culture was addedto each TK culture and cultures were grown for 48 hours at 37° C. in 5%CO₂. An aliquot of supernatant from each culture was harvestedimmediately after inoculation to establish a baseline density ofbacteria for each culture. After 48 hours, 2 ml of supernatant wereharvested from each culture as well as from uninfected TK cultures. Oneml of each supernatant was then analyzed by a spectrophotometer at anOD₆₀₀ to measure for bacterial density. Supernatants from uninfectedcultures were used as a blank to correct for background. As shown inFIG. 6, bacterial growth was significantly suppressed in TK-hPMS2-134(R1) and TK-hPMS2-134 (R2) cultures in contrast to TKvect control cells.These data demonstrate the feasibility of using a dominant-negativemismatch repair mutant hPMS2-134 on mammalian cells to producegenetically altered clones capable of producing a molecule(s) that cansuppress microbial growth.

As those skilled in the art will appreciate, numerous changes andmodifications may be made to the preferred embodiments of the inventionwithout departing from the spirit of the invention. It is intended thatall such variations fall within the scope of the invention. In addition,the entire disclosure of each publication cited herein is herebyincorporated herein by reference.

1-36. (canceled)
 37. A method for obtaining a genetically stablemammalian cell that is resistant to a selected microbe comprising:growing a culture of mammalian cells wherein said cells comprise adominant-negative allele of a mismatch repair gene, wherein saidmismatch repair gene encodes the first 133 amino acids of PMS2, andallowing mutations to occur; exposing said cells to said selectedmicrobe; selecting said mammalian cell that is resistant to saidselected microbe; and suppressing expression of said dominant negativeallele of said mismatch repair gene; thereby obtaining a geneticallystable mammalian cell that is resistant to a selected microbe.
 38. Themethod of claim 37 wherein said hypermutable cell is selected forresistance to a bacterium.
 39. The method of claim 37 wherein saidhypermutable cell is selected for resistance to a fungus.
 40. The methodof claim 37 wherein said dominant negative allele of said mismatchrepair gene is operably linked to an inducible promoter.
 41. The methodof claim 40 wherein expression of said dominant negative allele issuppressed by withdrawing an inducer of said inducible promoter.
 42. Themethod of claim 37 wherein expression of said dominant negative alleleis suppressed by knocking out said dominant negative allele.
 43. Themethod of claim 37 wherein said step of selecting for microbialresistance comprises isolating and testing culture medium from saidhypermutable cell.
 44. A method for obtaining a genetically stable cellcomprising a mutation in a gene encoding an antimicrobial activitycomprising: growing a culture of mammalian cells comprising said geneencoding said antimicrobial activity, and a dominant negative allele ofa mismatch repair gene, wherein said mismatch repair gene encodes thefirst 133 amino acids of PMS2, and allowing mutations to occur;selecting a cell comprising said antimicrobial activity; determiningwhether said gene comprises a mutation; and suppressing said dominantnegative allele of said mismatch repair gene; thereby obtaining agenetically stable cell comprising a mutation in a gene encoding anantimicrobial activity.
 45. The method of claim 44 wherein saidhypermutable cell is selected for resistance to a bacterium.
 46. Themethod of claim 44 wherein said hypermutable cell is selected forresistance to a fungus.
 47. The method of claim 44 wherein said step ofselecting a cell for antimicrobial activity comprises isolating andtesting culture medium from said hypermutable cell.
 48. The method ofclaim 44 wherein said dominant negative allele of said mismatch repairgene is operably linked to an inducible promoter.
 49. The method ofclaim 48 wherein expression of said dominant negative allele issuppressed by withdrawing an inducer of said inducible promoter.
 50. Themethod of claim 44 wherein expression of said dominant negative alleleis suppressed by knocking out said dominant negative allele.
 51. Amethod for obtaining an antimicrobial agent comprising: growing aculture of mammalian cells wherein said cells comprise adominant-negative allele of a mismatch repair gene, wherein saidmismatch repair gene is a PMS2 gene or homolog thereof that comprises amutation resulting in a reduced ability to interact with MLH1, andallowing mutations to occur; exposing said cells to said selectedmicrobe; selecting said mammalian cell that is resistant to saidselected microbe; culturing said selected mammalian cell that isresistant to said selected microbe; and purifying antimicrobial materialfrom said cultured mammalian cell; thereby obtaining an antimicrobialagent.
 52. The method of claim 51 wherein said antimicrobial agent ispurified from culture medium of said cultured, selected mammalian cellthat is resistant to said selected microbe.
 53. The method of claim 51wherein said mammalian cells are selected for resistance to a bacterium.54. The method of claim 51 wherein said mammalian cells are selected forresistance to a fungus.
 55. The method of claim 51 wherein said dominantnegative allele of said mismatch repair gene is operably linked to aninducible promoter.
 56. The method of claim 55 further comprising thestep of suppressing expression of said dominant negative allele of saidmismatch repair gene after selecting said mammalian cell that isresistant to said selected microbe.
 57. The method of claim 56 whereinexpression of said dominant negative allele is performed by withdrawingan inducer of said inducible promoter.
 58. The method of claim 51further comprising the step of suppressing expression of said dominantnegative allele of said mismatch repair gene after selecting saidmammalian cell that is resistant to said selected microbe by knockingout said dominant negative allele of said mismatch repair gene.